Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor

The translocation of fluorescently tagged PLC gamma and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC gamma to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P(2) substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The presence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.     

Mannose-binding lectin deficiency is associated with smaller infarction size and favorable outcome in ischemic stroke patients

Background

The Mannose-binding lectin (MBL) pathway of complement plays a pivotal role in the pathogenesis of ischemia/reperfusion (I/R) injury after experimental ischemic stroke. As comparable data in human ischemic stroke are limited, we investigated in more detail the association of MBL deficiency with infarction volume and functional outcome in a large cohort of patients receiving intravenous thrombolysis or conservative treatment.

Methodology/Principal Findings

In a post hoc analysis of a prospective cohort study, admission MBL concentrations were determined in 353 consecutive patients with an acute ischemic stroke of whom 287 and 66 patients received conservative and thrombolytic treatment, respectively. Stroke severity, infarction volume, and functional outcome were studied in relation to MBL concentrations at presentation to the emergency department. MBL levels on admission were not influenced by the time from symptom onset to presentation (p = 0.53). In the conservative treatment group patients with mild strokes at presentation, small infarction volumes or favorable outcomes after three months demonstrated 1.5 to 2.6-fold lower median MBL levels (p = 0.025, p = 0.0027 and p = 0.046, respectively) compared to patients with more severe strokes. Moreover, MBL deficient patients (<100 ng/ml) were subject to a considerably decreased risk of an unfavorable outcome three months after ischemic stroke (adjusted odds ratio 0.38, p<0.05) and showed smaller lesion volumes (mean size 0.6 vs. 18.4 ml, p = 0.0025). In contrast, no association of MBL concentration with infarction volume or functional outcome was found in the thrombolysis group. However, the small sample size limits the significance of this observation.

Conclusions

MBL deficiency is associated with smaller cerebral infarcts and favorable outcome in patients receiving conservative treatment. Our data suggest an important role of the lectin pathway in the pathophysiology of cerebral I/R injury and might pave the way for new therapeutic interventions.     

Copeptin as a Marker for Severity and Prognosis of Aneurysmal Subarachnoid Hemorrhage

Background

Grading of patients with aneurysmal subarachnoid hemorrhage (aSAH) is often confounded by seizure, hydrocephalus or sedation and the prediction of prognosis remains difficult. Recently, copeptin has been identified as a serum marker for outcomes in acute ischemic stroke and intracerebral hemorrhage (ICH). We investigated whether copeptin might serve as a marker for severity and prognosis in aSAH.

Methods

Eighteen consecutive patients with aSAH had plasma copeptin levels measured with a validated chemiluminescence sandwich immunoassay. The primary endpoint was the association of copeptin levels at admission with the World Federation of Neurological Surgeons (WFNS) grade score after resuscitation. Levels of copeptin were compared across clinical and radiological scores as well as between patients with ICH, intraventricular hemorrhage, hydrocephalus, vasospasm and ischemia.

Results

Copeptin levels were significantly associated with the severity of aSAH measured by WFNS grade (P = 0.006), the amount of subarachnoid blood (P = 0.03) and the occurrence of ICH (P = 0.02). There was also a trend between copeptin levels and functional clinical outcome at 6-months (P = 0.054). No other clinical outcomes showed any statistically significant association.

Conclusions

Copeptin may indicate clinical severity of the initial bleeding and may therefore help in guiding treatment decisions in the setting of aSAH. These initial results show that copeptin might also have prognostic value for clinical outcome in aSAH.     

High-speed AFM reveals the dynamics of single biomolecules at the nanometer scale

Atomic force microscopy allows visualization of biomolecules with nanometer resolution under physiological conditions. Recent advances have improved the time resolution of the technique from minutes to tens of milliseconds, meaning that it is now possible to watch single biomolecules in action in real time. Here, we review this development.     

The hypercholesterolemic effect of cafestol in coffee oil in gerbils and rats

Coffee beans contain the diterpene cafestol, which raises plasma cholesterol concentrations in humans. Daily consumption of 2 g coffee oil, which provides approximately 60 mg cafestol (equivalent to 5.7 mg cafestol/MJ), increases plasma cholesterol concentrations by 28%. We studied the effect of cafestol in coffee oil on gerbils and rats to determine whether the pathways that lead to cafestol-induced hypercholesterolemia in humans are also present in other species. We fed coffee oil from the same batch used in humans to female gerbils and rats. Gerbils were fed a semipurified diet containing 0.5% or 5% (w/w) coffee oil (equivalent to 8.7 and 86.8 mg cafestol/MJ, respectively) in the presence or absence of 0.05% (w/w) cholesterol for a period of 10 weeks. When compared with the gerbils fed no coffee oil, the addition of 0.5% coffee oil to the diets did not affect plasma cholesterol. Plasma cholesterol was significantly higher only when 5% coffee oil was fed, both in the absence (1.01 mmol/L, 33% higher) and presence (1.87 mmol/L, 70% higher) of dietary cholesterol. Liver weight was also significantly higher when 5% coffee oil was fed. Rats were also fed diets containing 0.5% or 5% coffee oil (equivalent to 8.7 and 86.8 mg cafestol/MJ) with and without 0.05% cholesterol for 8 weeks. Feeding 0.5% coffee oil compared with no coffee oil resulted in significantly higher plasma cholesterol levels throughout the study both in the absence (0.46 mmol/L, 27% higher) and presence (0.28 mmol/L, 15% higher) of dietary cholesterol. Diets containing 5% coffee oil appeared to be toxic. Thus, coffee oil diterpenes can result in higher plasma cholesterol in gerbils and rats. The failure to observe these effects in previous studies may be due to doses that were too low.     

Multiplexed FRET to image multiple signaling events in live cells

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.     

Characterization of Colletotrichum gloeosporioides isolates from avocado and almond fruits with molecular and pathogenicity tests.

One hundred twenty isolates of Colletotrichum gloeosporioides from avocado (6 U.S. and 57 Israeli isolates) and almond (57 Israeli isolates) fruits were compared by various molecular methods and a pathogenicity assay in order to determine the genetic diversity and host specificity between and among the different populations. DNA from eight additional U.S. almond anthracnose isolates were also compared. PCR amplification of genomic DNA with four primers produced uniform banding patterns for all the Israeli almond isolates from different geographic locations in Israel. DNAs from the U.S. almond isolates were distinct from DNAs of the Israeli isolates. In contrast, the avocado isolates from Israel and the United States were more diverse, with numerous arbitrarily primed-PCR phenotypes being observed. HaeIII digestion patterns of A+T-rich DNA distinguished between the almond and avocado isolates. Southern hybridization of the repetitive nuclear-DNA element GcpR1 to PstI-digested genomic DNA of almond and avocado isolates revealed no polymorphic fragments among the almond isolates, whereas polymorphic fragments were observed among the avocado isolates. Amplification and subsequent restriction enzyme digestion of the internal transcribed spacer 4 and 5 regions between the small and large nuclear subunits of DNA encoding rRNA failed to distinguish between C. gloeosporioides isolates from a diverse host range. In artificial inoculations, avocado isolates produced various lesions on avocado and almond fruits, whereas the almond isolates infected both fruits at a lower rate.     

Controlling the seed bank of the invasive plant <Emphasis Type="Italic">Acacia saligna</Emphasis>: comparison of the efficacy of prescribed burning,soil solarization,and their combination

Reducing the seed bank of invasive plants is a prerequisite for successful restoration of invaded ecosystems. The Australian legume tree Acacia saligna is one of the worst invasive plants in Mediterranean climate regions. This fire-adapted species possesses a large persistent seed bank characterized by physical dormant seeds. The present research was aimed to compare the efficacy of prescribed burning, soil solarization by solar heating of moist soil, and their use in combination on A. saligna seed bank reduction, using the buried seed method, and on seedling emergence from the natural seed bank, as a model of controlling fire-adapted plants. The data obtained show that the direct effect of prescribed burning on the buried seed dynamics was highly variable, and it only reduced seed viability from 98% to about 56%. Soil solarization, particularly in combination with prescribed burning, was much more effective than prescribed burning alone, reducing seed viability to about 29 and 4%, respectively. These results were confirmed by recording seedling emergence from the natural seed bank during two successive germination years following the treatments. Only a relatively very small number of seedlings emerged in the soil solarization treatment and none in the combined treatment. Based on the above data, it is recommended to apply prescribed burning as a pretreatment for soil solarization, or to utilize wild fires followed by soil solarization to reduce the seed bank of invasive fire-adapted plants. In situations in which fire cannot be used as a pretreatment, soil solarization alone is reasonably effective.